Charcoal dextran stripped human serum

MCF-7 cells were cultured in 6-well plates at a final concentration of 4 × 10⁵ cells per well in RPMI medium without phenol red (Gibco BRL, Grand Island, NY, USA) with 100 μg/mL streptomycin, 100 U/mL penicillin, and 5% charcoal-dextran stripped human serum (Innovative Research, Novi, MI, USA) for 24 h. MCF-7 cells were treated with compound 4 (25 μM to 100 μM) for 24 h. Proteins (20 µg) from MCF-7 cells were fractionated by SDS-PAGE (10% polyacrylamide gel) and transferred onto a polyvinylidene fluoride (PVDF) membrane. Phospho-extracellular signal-regulated kinase (p-ERK), ERK, phospho-phosphatidylinositol 3-kinase (p-PI3K), PI3K, phospho-Akt, Akt, phospho-estrogen receptor α (p-ERα), ERα, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling Technology) were used as primary antibodies. Protein bands were visualized on a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany) using ECL Advance Western blotting detection reagents (GE Healthcare, Little Chalfont, UK).

The Ez-Cytox cell viability assay kit was obtained from the Daeil Lab Service Co. (Seoul, Korea). RIPA buffer, primary antibodies against BH3-interacting domain (BID), Bax, Bcl-2, cleaved caspase-8, cleaved caspase-3, cleaved caspase-9, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), poly ADP ribose polymerase (PARP), and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibodies were obtained from Cell Signaling (Danvers, MA, USA). The Pierce™ BCA Protein Assay Kit was obtained from Thermo Scientific (Waltham, MA, USA). RPMI1640 medium was purchased from Cellgro (Manassas, VA, USA). Fetal bovine serum (FBS) and phenol-red-free RPMI medium were obtained from Gibco BRL (Grand Island, NY, USA). Charcoal-dextran-stripped human serum was purchased from Innovative Research (Novi, MI, USA). ECL Advance Western blotting detection reagents were obtained from GE Healthcare (Little Chalfont, UK).

The E-screen assay reflects an increase in proliferation rates after treatment of the test substance [21 (link)]. In our study, the proliferation rates of MCF cells were measured using the EZ-Cytox assay kit (Daeil Lab Service Co., Seoul, Korea). This kit measures cellular mitochondrial activity upon the conversion of water-soluble tetrazolium salt (WST-1) to insoluble formazan crystals [32 (link),33 (link)]. MCF-7 cells were seeded in 24-well plates (1 × 10⁵ cells per well) in a phenol red-free RPMI medium (Gibco BRL, Grand Island, NY, USA) supplemented with an antibiotic solution for 24 h. Charcoal-dextran-stripped human serum at 5% (Innovative Research, Novi, MI, USA) was added to remove estrogen in serum [34 (link),35 (link)]. MCF-7 cells were treated with concentrations of 5–100 μM gentiopicroside, macelignan, γ-mangostin, three lignans (schisandrol A, schisandrol B, and schisandrin C), and E2 for 144 h, either with or without 100 nM ICI 182,780 (ICI), an ER antagonist [36 (link),37 (link)]. Then, the cells were incubated with Ez-Cytox reagents for 40 min, and the absorbance of the reaction product was measured at 450 nm using a microplate reader (PowerWave XS, Bio Tek Instruments, Winooski, VT, USA).